The goal of this Phase l project is to isolate a temperature sensitive mutant of streptavidin that will bind biotin at 4C but not at 4OC. This will be accomplished by chemical mutagenesis to saturate the streptavidin gene with mutations. A scheme for enriching for the temperature sensitive mutant will be employed. The library of mutants and the enriched library will both be screened using a computer assisted system for colony screening to find candidate mutants. These mutants will be characterized and the resulting mutants are expected to provide a valuable and novel addition to avidin-biotin technology. PROPOSED COMMERCIAL APPLICATION: This project will result in the isolation of a novel form of streptavidin that will allow for the conditional binding of biotynlated substrates. This will be particularly useful in affinity purification applications, isolation of specific antibody producing cells for production of monoclonal antibodies, and isolation of specific subsets of cells from mixed populations, such as blood progenitor cells to he used in autografts after chemotherapy in cancer patients.